Revised Abstract: Rhabdomyosarcoma (RMS) is the third most common soft tissue sarcoma in children. Alveolar RMS is characterized by the t(2;13) that results in the expression of the activated transcription factor Pax3-FKHR. The long-term goal of this project is to identify the tumorigenic events that result in alveolar RMS. Because Pax3 plays an important role in the embryonic development of muscles, the proposed hypothesis is that disruption of Pax3-regulated gene expression by Pax3-FKHR is crucial for the development of alveolar RMS. However, expression of Pax3- FKHR in mice is insufficient to recapitulate disease. Forced expression of Pax3 and Pax3-FKHR in primary mouse myoblasts increases their rate of proliferation, and Pax3-FKHR, but not Pax3, blocks their differentiation. Therefore, the following specific aims are proposed: Aim 1) The mechanism by which Pax3-FKHR causes the acceleration of the cell cycle in primary myoblasts and the transcriptional targets of Pax3 and Pax3-FKHR in myoblasts will be identified. This aim will be accomplished by analyzing the interactions between Pax3-FKHR and cell-cycle regulators and by performing cDNA microarray analyses of myoblasts. Aim 2) The genes that cooperate with Pax3-FKHR in transforming primary myogenic in vitro and in vivo will be identified. Complementary mutagenesis approaches will be used to identify the additional genetic changes that are essential for tumorigenic transformation. Such changes will be introduced into mouse models to test their ability to cooperate with Pax3-FKHR in tumorigenesis. The relevance of such changes in human alveolar RMS will be determined by testing for disease-specific changes in expression of these genes in human RMS cell lines and tumors. Together, these studies will provide valuable insights into the disruption of molecular pathways that cause alveolar RMS in children. This understanding may ultimately result in the identification of new targets that will lead to improved therapies for this disease.